Stable Nanomagnetic Particle Dispersions

ABSTRACT

Processes and compositions are described for preparing new, colloidally stable, coated nanomagnetic particles useful for both in-vitro and in-vivo biomedical applications, including cell targeting and capturing cells, microorganisms, and cellular organelles or entities such as exosomes. These nanomagnetic particles can also be used as imaging contrast agents due to their small size and high magnetic moment. The nanomagnetic particles include a series of sequentially added, stabilizing surface coatings rendered onto nano-sized magnetic crystal clusters (e.g., magnetite particles) to impart colloidal stability in complex biological samples with minimal leaching of the coating materials, high binding capacity, and low non-specific binding. Another benefit of this invention is the ability to utilize both external and internal magnetic field-generating separation devices to effect separation of the magnetic nanoparticles.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/143,552, filed on Apr. 30, 2016, entitled STABLE NANOMAGNETICPARTICLE DISPERSIONS, naming Dhanesh Gohel, Hong Zhang, and John Ransomas inventors, and designated by Attorney Docket No. BLD-0010-UT, whichclaims the benefit of U.S. Provisional Patent Application No.62/156,141, filed on May 1, 2015, entitled STABLE NANOMAGNETIC PARTICLEDISPERSIONS, naming Dhanesh Gohel, Hong Zhang, and John Ransom asinventors, and designated by Attorney Docket No. BLD-0010-PV. The entirecontent of the foregoing patent applications is incorporated herein byreference, including all text, tables and drawings.

BACKGROUND OF THE INVENTION

Magnetic particle-based technologies for the separation and isolation ofcells, nucleic acids, proteins, and other biomolecules have becomeestablished and improved over the past several decades. Magneticparticles are typically conjugated with specific targeting moieties suchas antibodies or nucleic acids, allowing the particles to bind to thetarget molecules found in complex mixtures such as cell populations orprotein and nucleic acid mixtures. The magnetic particles bound to thetarget biological material can then be separated from the mixture usingmagnetic field devices, providing a purification or enrichment methodfor the target. Such magnetic particle-based biological target isolationapproaches have been used to isolate or enrich eukaryotic cells bearingtarget antigens, bacterial species, nucleic acids, and proteins. Theyhave also been used in clinical testing applications such as serving assolid supports for immunoassays or radioimmunoassays (RIA).

Methods for preparing magnetic particles for such applications aretypically of two general types. One general method involves dispersingthe magnetic particles evenly within a polymeric matrix duringpreparation of the polymeric particles, constructing a magnetic materialshell around a polymeric particle core, or introducing magnetic materialinto pre-existing pores within the polymer particles. Examples of theformer method can be found, for example, in U.S. Pat. No. 4,358,388, andof the second method in U.S. Pat. Nos. 5,320,944 and 5,091,206. Thelatter method is exemplified in U.S. Pat. Nos. 5,648,124 and 4,654,267.All of these methods result in magnetic particles of greater than 0.3 μm(micrometer) in size.

The second general method for preparing magnetic particles forbiomaterial applications involves creating bare magnetic materialparticles first that serve as the core of a larger particle created byconstructing a shell around the first magnetic material core. One formof primary coating has been a silane coat, but other coatings have alsobeen described. For example, U.S. Pat. No. 3,933,997 describes the useof a silane coupling agent that coats magnetic particles and directlyconjugates to specific antibodies. This material was reportedly intendedfor use in RIA methods. U.S. Pat. No. 4,554,088 describes constructionof a metal or iron oxide particle core that is coated by a polymericsilane to which bioaffinity molecules such as antibodies are directlycoupled. U.S. Pat. No. 4,695,392, a division of the aforementioned '088patent, further defines the silane coat to which bioaffinity moleculesare directly attached as having two discrete functionalities—the firstto adsorptively or covalently couple to the metal oxide core particleand the second to covalently couple to bioaffinity organic molecules. Inboth patents the size of particles is defined as ranging from 0.1 um to1.5 um. U.S. patent application publication no. 2007/0026435, nowabandoned, discloses a hydroxysilane, preferablyhydroxyalkyltrialkoxysilane, primary coating on a magnetic particlecore. In this application the particle sizes ranged from 0.1 um to 100um, and the particles were specified for use in isolation of specificnucleic acids from mixtures. The magnetic particles disclosed in boththe '392 patent and the 2007/0026435 publication produce highlyaggregated magnetic particles upwards of 1 μm in diameter when strictlyadhering to the cited examples contained therein. U.S. Pat. No.7,169,618 discloses preparation of magnetic particles of a size rangefrom 0.07 um to 0.45 um that are first coated with an organosilane thatis then conjugated with a polysaccharide material via a pendantfunctional group on the organosilane. U.S. patent applicationpublication no. 2010/0012880 discloses a magnetic particle having amagnetic material core with a primary hydrophobic protective layer overwhich is layered a hydrophilic alkylsilane coating. Such particles aredisclosed as being from 0.2 um to 0.4 um in diameter.

Distinct from silane coatings that also serve as the coupling reagent tobioaffinity molecules, non-silane primary coatings on core magneticparticles have also been reported. These include polyglutaraldehyde(see, e.g., U.S. Pat. No. 4,267,234), acrylamide, n-butylacrylate, orN,N′-methylenebisacrylamide (see, e.g., U.S. Pat. No. 4,454,234),polyacrolein (see, e.g., U.S. Pat. No. 4,783,336), polyvinyl alcohol(see, e.g., U.S. Pat. No. 6,204,033), natural polymers like dextran(see, e.g., U.S. Pat. No. 4,452,773), and bovine serum albumin (see,e.g., U.S. Pat. No. 4,795,698). All of these magnetic particle primarycoatings reportedly serve as substrates to which additional biomoleculessuch as antibodies or nucleic acids may be conjugated. With all of thesemethods, the shapes and sizes of the resultant bioaffinity magneticparticle products are not easily controlled, the size range of theparticle products are relatively broad, the diameters are typicallygreater than 0.5 um, and the product particles tend to easily adhere toone another forming particle clumps.

Despite these advances, the need exists for further improved magneticparticles, as well as processes for making and using such particles.

SUMMARY OF THE INVENTION

This invention addresses these needs, and provides a highly reproducibleprocess for producing silane—(glass) encapsulated nanomagnetic particlesonto which is further encapsulated a stabilizing protein/polymercomposite mixture to result in resuspendable nanomagnetic particles thatcan withstand multiple rounds of exposure to strong magnetic fields(e.g., 0.5-1.0 Tesla) without any substantial increase in particle size.Such multi-layered nanomagnetic particles are then made specific for oneor more desired biomolecule species, cell, or tissue type by covalentlyattaching targeting moieties to the protein/polymer composite layer. Theresultant nanomagnetic particles additionally have a very narrow sizedistribution with a polydispersity index (PDI) value 0.10) approachingthat of monodisperse particles. Nanomagnetic particles of diameters fromabout 5 nm to about 500 nm can be produced using this invention,preferably from about 30 nm to about 300 nm. Preferred nanoparticles ofthe invention include a magnetic core particle comprised of a ferrousoxide, particularly magnetite (Fe₃O₄) crystal clusters. Other preferredmagnetic cores comprise Fe₂O₃; a chromium oxide, for example, CrO₃; or astable metal oxide that comprises a substituted metal ion, e.g., Mn, Co,Ni, Zn, Gd, and Dy. Particularly preferred magnetic core particles,including those comprised of magnetite crystal clusters, have diametersranging from about 5 nm to about 300 nm.

The nanomagnetic particles so produced have three layers of coatingsaround the core nano-sized magnetic particles, namely a silane or glasslayer, a protein/polymer layer, and finally an outermost layer that iscomprised of targeting moieties, which are one member of a bioaffinityligand pair, such as an antibody for targeting an antigen of interest, acell surface receptor or receptor fragment, etc. The targeting moiety orbioaffinity ligand (which may be, for example, an antibody orantigen-binding antibody fragment, streptavidin, peptide, nucleic acidpolymer, or other receptor or ligand of interest) is preferablycovalently conjugated to the ample functional groups present on theprotein/polymer layer. In preferred embodiments, the glass layer is asilane layer formed from organofunctional alkoxysilane molecules,optionally organofunctional alkoxysilane molecules that comprise acouplable end group, optionally a couplable end group selected from thegroup consisting of an amino, sulphydryl, carboxyl, and hydroxyl end orreactive group. The end group may be protected or unprotected; ifprotected, a deprotection step is preferably used prior to coupling ofthe protein/polymer composite layer. In preferred embodiments, theprotein/polymer composite layer is covalently bound to the glass layer.Preferably, the protein/polymer composite layer is comprised of serumalbumin, e.g., bovine or human serum albumin, dextran, or casein. Insome embodiments, the protein/polymer composite layer is permanentlybound by heating the composition from about 45° C. to about 85° C. Thetargeting moiety or bioaffinity ligand (i.e., one member of a highaffinity binding pair) is then conjugated, preferably covalently, to theprotein/polymer layer. Preferred targeting moieties include antibodies(preferably monoclonal antibodies), antigen-binding antibody fragments(e.g., Fab fragments), cell surface receptors, ligand-bindingextracellular domains of cell surface receptors, nucleic acids(including nucleic acid-based aptamers), avidin, streptavidin, biotin,and pharmaceutical compounds for purposes of targeted drug delivery.

The targeted nanomagnetic particles of the invention behave as stablecolloids when combined in a reaction mixture with complex liquids, forexample, mammalian whole blood or a fraction of mammalian whole blood.Moreover, targeted nanomagnetic particles of the invention preferablyexhibit no significant or deleterious change in magnetic, bioaffinity,and/or particle size and targeting properties during storage over longperiods, e.g., 1 year to 5 years. Preferred sources of biologicalsamples are those obtained from mammals, including humans, as well asfrom companion animals (e.g., cats and dogs) or those of commercialsignificance (e.g., cattle; fowl such as chickens, turkeys, and ducks;goats; horses, pigs, sheep, etc.).

Compositions comprising the nanomagnetic particles of the invention canbe formulated in any suitable manner, including dry, readily dispersibleformulations (e.g., lyophilized formulations) or liquid compositions.After preparation, such compositions are typically dispensed in desiredquantities (e.g., in an amount suitable for performing a single magneticseparation, or alternatively, multiple separations) into suitablecontainers that are then often packaged into kits for subsequentdistribution and use. Kits according to the invention preferably includeinstructions for use of the reagents in the kit, including use ofnanomagnetic particles of the invention to perform one or more desiredmagnetic separations. In some embodiments, such kits may include aplurality of targeted nanomagnetic particle species, wherein eachtargeted nanomagnetic particle species comprises a different targetingmoiety species. Preferably, in kits that contain a plurality ofdifferent targeted nanomagnetic particle species, each species ispreferably packaged in a separate container in the kit. In someembodiments, such kits may also include compositions for also performingbuoyant separations of one or more particular biomolecule species from areaction mixture prepared from a biological sample.

Thus, this invention relates to the use of magnetic separation toseparate target biomolecules, for example, cells, organelles, exosomes,oncosomes and other biological materials to be isolated or separatedfrom complex mixtures such as biological samples. To accomplish suchseparations, this invention provides a new class of patentablenanomagnetic particle compositions for use in magnetic separationprocedures.

These and other aspects, objects, and embodiments of the presentinvention, which are not limited to or by the information in thisSummary, are provided below, including in the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an acid dissolution study performed onnon-silanized silanized and silanized nanomagnetic particles of theinvention pre- and post-sonication. The plot shows the percentage ofiron dissolved after a 15 min. exposure to 4 M HCl.

FIG. 2 shows the magnetic separation efficiency of various nanomagneticparticles made according to this invention.

FIG. 3 shows the magnetic separation efficiency of anantibody-conjugated commercially available nanobead product.

FIG. 4 shows the purity of CD4 positive cells that were negativelyselected using Streptavidin-conjugated nanomagnetic particles andappropriate biotinylated antibodies that were stored at varioustemperatures and then tested for cell separation performance over thecourse of two months.

FIG. 5 shows the CD4 positive cell yield of Streptavidin-conjugatednanomagnetic particles that were stored at various temperatures andtested for cell separation performance as in FIG. 4 over the course oftwo months.

FIG. 6 shows the purity of rat anti-mouse CD19 antibody-conjugatednanomagnetic particles that were stored at various temperatures andtested for CD19 positive cell separation performance over the course oftwo months.

FIG. 7 shows the yield of rat anti-mouse CD19 antibody-conjugatednanomagnetic particles that were stored at various temperatures andtested for CD19 positive cell separation performance over the course oftwo months.

FIG. 8 is a plot showing the particle size distributions of variousconventional, commercially available magnetic particles compared tothose of the invention produced in accordance with Example 1, below.Measurements were made using dynamic light scattering and the percentageof particles in various ‘size-bins’ was plotted as a function of actualparticle size.

FIG. 9 has two panels, A and B, each of which contain 3 transmissionelectron micrographs. The micrographs in Panel A show cells magneticallyselected by HGMS using commercially HGMS compatible magnetic particles,while the micrographs in Panel B show cells magnetically selected byHGMS using targeted nanomagnetic particles of the present invention.

FIG. 10 shows a plot of relative fluorescence units (RFU) versusconcentration of anti-mouse CD3 antibody used for coating microwells todrive the cells to proliferate. RFU is an index of the relative numberof cells in each condition.

FIG. 11 illustrates the general scheme for “magnetibuoyant” separationmethods of the invention.

FIG. 12 illustrates the principle of using microbubbles to isolatespecific cells.

FIG. 13 illustrates the general scheme for “magnetibuoyant” rare cellseparation methods of the invention.

FIG. 14 illustrates the isolation of human CD4+ lymphocytes from acomplex mixture.

DETAILED DESCRIPTION

As those in the art will appreciate, the following detailed descriptiondescribes certain preferred embodiments of the invention in detail, andis thus only representative and does not depict the actual scope of theinvention. Before describing the present invention in detail, it isunderstood that the invention is not limited to the particular aspectsand embodiments described, as these may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention defined by the appended claims.

Numerous methods are known for analyzing and sorting populations ofcells and other biomolecules, including methods based on cell size,density, or granularity in which separation is achieved bysedimentation, alone or in combination with density gradients andcentrifugation or elution. Other methods include those based ondifferential resistance of cells to osmotic lysis, as can be used, forexample, to separate white blood cells from whole blood. Furthermore,methods of depleting (i.e., reducing the number of) unwanted cells (orother biomolecules) from a more complex biological sample using specificantibodies that react with a cell surface marker can be used to removeor reduce the numbers of cells expressing that marker. Still other cellseparation methods include flow cytometry and magnetic cell sorting(e.g., using magnetic particle-conjugated antibodies), as well as othermethods that employ antibody affinity (or other high affinity bindingpairs) to particular biomolecules, including cell surface proteins.Using these technologies, positive enrichment or depletion ofparticularly desired, i.e., “targeted” or “target”, cell populations(i.e., those expressing a marker that can be targeted by the highaffinity binding moiety (e.g., an antibody, Fab fragment, receptor,etc.) conjugated to the labeled detection/separation reagent can beachieved.

Thus, this invention addresses the separation of one or more desired ortarget biomolecule species, particularly one or more target cellpopulations, from a more complex biological sample such as a cellularmixture (e.g., whole blood, a homogenized biopsy or tissue sample,etc.). A “target biological material” or “target biomolecule” refers toany biological substrate, for example, cells, organelles, and otherbiological materials, a user desires to isolate, enrich for, deplete, ortarget and for which a specific binding moiety (partner) can be preparedso as to specifically label or bind the material. The list of suitabletarget biomolecules is extensive, and includes microorganisms such asprotozoa, bacteria, yeast, and other fungi, cultured cells frommulti-celled organisms (including mammalian and other vertebrate cells,viruses, and fragments of the cells and viruses), eukaryotic cellpopulations that express one or more targetable cell surface antigens,and organelles or other subcellular structures (e.g., exosomes,proteasomes, ribosomes, etc.) that include a targetable protein or otherbiomolecule (e.g., a carbohydrate, lipid, etc.). Indeed, any biologicalmaterial (i.e., biomolecule), either a single molecule (e.g., a protein)or an organized or amorphous aggregate of one or more molecules (of thesame of different molecular species), that can be targeted by atargeting moiety can be isolated or purified using the nanomagneticparticles and methods of the invention.

The instant methods are based on the use of the new patentable class oftargeted nanomagnetic particles described herein, which can be used toseparate targeted biomolecules (up to and including intact, viablecells) from other components in a reaction mixture by magnetic cellseparation techniques. If desired, other separations can also beperformed in order to enrich or deplete one or more other biomoleculespecies (e.g., cell populations) present in the reaction mixture (as aresult of being present in the original sample to be analyzed). Indeed,in a related aspect, targeted biomolecular separation based on the useof targeted buoyant microparticles (e.g., microbubbles and the like) canbe used in conjunction with magnetic separation for parallel or serialprocessing of a biological sample in order to enrich for two or moredesired cell populations (or other biomolecule species) or to enrich forat least one target cell population (or other biomolecule species) anddeplete another. Particularly preferred proteins that can be targeted,for example, by a monoclonal antibody specifically reactive therewith,to separate target cell populations from biological samples include thefollowing cell surface proteins:

Human Specificity Mouse Specificity CD4 CD4 CD8 CD8 CD19 CD19 CD14 CD11cCD56 CD25 CD25 Ter119 CD235 CX3CR1 Epcam/CD326 CD20 TSPAN33 CD20 Lfr5ERBB2/HER2 GPR35/CXCR8

In the context of the invention, targeted separation (for enrichment ordepletion) is achieved through the use of a targeting moiety conjugatedto the separable particle (e.g., a nanomagnetic particle of theinvention, a conventional magnetic particle, a buoyant particle (e.g., amicrobubble), etc.). The targeting moiety is typically a high affinitybinding reagent that can conjugated to the separable particle by asuitable chemistry (preferably one involving covalent bonding that doesnot disrupt binding between the high affinity binding reagent and thetargeted biomolecule, preferably a protein expressed on the surface of atargeted cell population, organelle, or other biomolecule). Examples ofsuch high affinity binding reagents include members of high affinitybinding pairs. Such members include antibodies (particularly monoclonalantibodies), antigen-binding antibody fragments (e.g., Fab fragments),or another member of a high affinity bending pair (one of which isconjugated to the separable particle and the other of which is the“target” present on the biomolecule or structure being targeted). Insome embodiments, the high affinity binding reagent and/or separableparticle to which it is conjugated is labeled with a detectable agentsuitable for cell separation (e.g., FACS), such as a fluorescent dye.

High affinity binding reagents conjugated to separable (e.g., bymagnetic or electric fields, buoyancy, etc.) particles can be used toseparate desired biomolecules (e.g., a cell population expressing aparticular cell surface antigen) from other reaction mixture componentsunder conditions that allow the binding reagents to specifically bindtheir corresponding targets (e.g., antigens in the case of antibodies,antigen-binding antibody fragments, etc.).

The practice of the separation methods of the invention comprise thefollowing steps: in a reaction mixture, immobilizing the targetbiomolecule, for example, a target cell population expressing aparticular cell surface marker, present in a biological sample known orsuspected to contain the target biomolecule, which biomolecule isspecifically bound by the targeting moiety of a nanomagnetic particle ofthe invention in a ferromagnetic matrix through the use of a magneticfield; washing the matrix to remove unbound components in the reactionmixture; and removing the magnetic field to elute the targetedbiomolecule from the matrix. As a result, a target biomolecule (e.g., atarget cell population) is enriched; in addition or alternatively, thebiological sample is depleted of the target biomolecule (provided thatat the material washed from the matrix is retained for further use).Elution of material from the ferromagnetic matrix can be performed usinggravity flow, centrifugation, vacuum filtration, or a pressure gradient.

The term “magnetic separation” refers to separation procedures forconstituent components of complex samples, e.g., biological samples.Such procedures include magnetic separation mediated by targetingmoieties that comprise one member of a high affinity binding pair (e.g.,a monoclonal antibody that specifically binds a target cell surfaceantigen) conjugated or otherwise linked to a nanomagnetic particleaccording to the invention. Magnetic separation can be combined withother separation procedures, including those that employ targetedbuoyant particles and/or separation techniques known in the art thatalso rely on high affinity binding pairs (e.g., antibodies and theircognate antigens), for instance, affinity chromatography, “panning”(where one member of the high affinity binding pair is attached to asolid matrix (e.g., the well of a microtiter plate). Fluorescenceactivated cell sorting (FACS) can also be used if fluorescent tags areincluded in the targeted separable particles. Indeed, any now known orlater developed ligand-dependent separation technique can be used inconjunction with positive and/or negative separation techniques thatrely on physical properties of the target biomolecule rather thanaffinity, including filtration, size exclusion chromatography, anddensity gradient centrifugation.

The invention also includes kits for performing the magnetic separationmethods described herein, alone or in addition to other separationmethods. Such kits include targeted nanomagnetic particles of theinvention that target a desired biomolecule, for example, a cell surfaceantigen expressed on the surface of a particular cell type. The targetednanomagnetic particles are typically packaged in containers that includesuch quantities of the particles as are needed to perform one or moremagnetic separation procedures. Instructions (or a link or websiteaddress containing such instructions) for use of the targetednanomagnetic particles (and any other included reagent(s), e.g.,targeted buoyant microbubbles) are also typically included in any suchkit.

Magnetic Separation

Among techniques known for separating components of a biologicalmaterial or sample are those that make use of magnetic separationtechniques. Magnetic separation methods typically selectively retainmagnetic materials in a chamber or column disposed in a magnetic field.Such methods typically include passing a biological material or samplethrough one or more separation columns. Briefly, the biological materialor sample is magnetically labeled by attachment to targeted nanomagneticparticles of the invention through the use of a targeting moietyconjugated to the particles, which targeting moiety targets a desired(or “target”) biomolecule known or suspected to be present in thesample, for example, displayed on the surface of certain cells known orsuspected to be present in the same. A suspension of the labeled targetsample is then applied to the separation chamber or column. To separatethe targeted biomolecule species from the remainder of the reactionmixture, the targeted biological material is retained in the chamber inthe presence of a magnetic field. The retained targeted biologicalmaterial can then be eluted by changing the strength of, or byeliminating, the magnetic field.

In some embodiments, high gradient magnetic separation (HGMS) is used(Miltenyi et al., Cytometry, 11, 231 (1990)). In HGMS, a matrix ofmaterial of suitable magnetic susceptibility such as iron wool or steelbeads is placed in a chamber or column such that when a magnetic fieldis applied, a high magnetic field gradient is locally induced close tothe surface of the matrix, permitting retention of complexes of themagnetized particles and targeted biological material formed through theassociation of the members of the high affinity binding pairs present inthe mixture.

The targeted nanomagnetic particles and methods of the invention can beused for the magnetic separation of, or to magnetically label and, ifdesired, isolate, any desired target substance or analyte (e.g., cells,organelles, etc.). Of particular interest is separating a specificbiomolecule from a complex biological mixture. The present invention hasgreat utility, in that almost any target substance may be separated oncea specific binding member for that substance is available. The targetingmoiety can be any member of a specific, high-affinity binding pair, or asubstance associated with a member of a specific, high-affinity bindingpair. For example, a cell surface antigen-antibody binding pair can beused to isolate the antigen itself, cells that express the antigen, aparticular organelle involved in processing of the antigen, etc. Thedevices and methods of the present invention are also advantageouslyapplied to diagnostic techniques involving the binding of a receptor andligand, such as immunoassays, and the like.

Targeted Nanomagnetic Particles

Two classes of magnetic oxides, ferrites and non-ferrites, can be usedfor the production of the targeted nanomagnetic particles of theinvention. Ferrites, or iron-containing transition metal oxides, cangenerally be represented as XO.Fe₂O₃, where “X” may be Fe, Ni, Cr, Co,Mn, Mg, Mo, Gd, Cu, V, Dy, Ey, Tm, or Yb. Therefore, in the process ofsynthesizing magnetite superclusters, one would substitute theFe²⁺-containing iron salt with one of the aforementioned divalent metalion salts. The most preferable in this class of ferrites is FeO.Fe₂O₃,which is better known as magnetite or Fe₃O₄. The non-ferrite class ofmagnetic oxides are void of the iron atom but instead are substitutedwith a combination of two or more ions of these transition metals: Cr;Co; Mn; Ni; Mo; Gd; Dy; Ey; Tm; and Yb. Such non-ferrite-based magneticoxides typically produce a spectrum of colored nanomagnetic particlesbut are less magnetically responsive than the ferrite class of magneticoxides.

Magnetite crystals were first synthesized almost a century ago. Thesubsequent processing and stabilization of the magnetite crystals hasspawned many different types of magnetic particles of different sizes,with different surface coatings, and for different applications. Inpreferred embodiments, magnetite (Fe₃O₄) crystals are first synthesizedusing any suitable process, including the well-known aqueous basedco-precipitation method [Massart 1982, Schwertmann 1991]. Stoichiometricmixtures of ferrous (Fe²⁺) and ferric (Fe³⁺) iron salts are titratedwith a strong base under an inert atmosphere to yield 1 um-3 μm diametermagnetite crystals. Variables such as the mole-ratio of the iron salts(e.g., 1.0 M Fe²⁺: 2.0 MFe³⁺ to 2.0 M Fe²⁺: 1.0 MFe³⁺), reactiontemperature (e.g., 40° C. to 95° C.), type of base counterions (e.g.,ammonium, sodium, potassium) used, and the rate of base addition (e.g.,2 mL/minute to 200 mL/minute) are optimized in order to produce thehighest quality ‘bare’ magnetite crystals. These magnetite crystals arenext sonicated at high power in order to yield quasi-stable 90 nm-110 nm(nanometer) sized nanomagnetic particles that are immediately silanizedusing an aqueous acidic silanization procedure concomitant with hightemperature dehydration in order to obtain silanized nanomagneticparticles.

Silanization can be accomplished using any suitable process. Forexample, after 25 minutes of sonication at high power (750W) using a 0.5inch titanium probe tip, nanomagnetic particles are transferred into a3-neck round-bottom glass reaction vessel kept under nitrogen gascontaining 50 v % glycerol together with an overhead stirrer. A 10 wt %(relative to the iron mass) solution of sodium silicate is then added at0.5 ml/minute, followed immediately by the addition of 0.5 M glacialacetic acid at 1 mL/minute until a pH of 6 is attained. The temperatureis then raised to 180° C. and the mixture is allowed to dehydrate for atleast 2 hours, then cooled and washed using water.

In various preferred embodiments, the ‘bare’ magnetite crystals arefirst peptized using a strong metal ion chelating agent such as EGTA inorder to make available additional seed hydroxyl groups for condensationwith the silanization reagent. Peptization is achieved by sonicating the2 um size magnetite superclusters in the presence of the chelating agent(e.g., EGTA) in order to introduce additional hydroxyl groups onto themagnetite particles and afford greater colloidal stability. In yetanother preferred embodiment, two silanization reagents are usedsequentially in order to both enhance encapsulation as well as toprovide additional couplable groups by virtue of the inherentfunctionalities present in the secondary silanization reagent.Sequential silanization can be achieved, for example, by firstsilanizing sonicated magnetite particles using sodium silicate asdescribed above, followed immediately by the addition of an amino-silanesuch as aminopropyl-trimethoxysilane (APTS) prior to dehydration at 180°C. (see Example 2, below).

A second round of high power sonication, albeit brief, is performed inorder to reduce the particle size, preferentially to 95 nm-105 nm. Next,these silanized nanomagnetic particles are mixed with a heated solutioncontaining a protein/polymer mixture, for example, BSA (bovine serumalbumin) and the polysaccharide dextran (99 wt % BSA: 1 wt % dextran to50 wt % BSA : 50 wt % Dextran). This can be accomplished, for example,by heating a solution containing a mixture of BSA and Dextran to 70° C.just prior to mixing it with sonicated magnetite particles in a sealed3-neck reaction vessel under a nitrogen atmosphere. The coating processis allowed to proceed for 30 minutes. The suspension then is cooled andwashed using, for example, a high-field magnetic dipole separator.

Heating concentrated BSA solutions to temperatures in excess of 58° C.is known to produce irreversible aggregates of BSA mediated by theformation of disulphide bonds and hydrogen bonding of beta sheetsbetween individual BSA molecules [Wetzel, 1980]. In one preferredembodiment, maleimide groups are introduced into the BSA protein priorto mixing with the sonicated silanized particles in order to furtherpromote the formation of disulphide bonds. The BSA/Dextran coatednanomagnetic particles are then washed with the aid of strongdipole/quadrupole-type magnetic separators to remove excess coatingmaterials as well as to narrow the size distribution of the particles toa final size of about 110 nm. The initial wash supernatant from thismagnetic fractionation step contains a significant amount (˜50% by ironmass) of 30 nm-80 nm size BSA/Dextran coated nanomagnetic particles.Such smaller sized nanomagnetic particles can also be effectivelyutilized for magnetically capturing/purifying intracellular and/orextracellular targets such as, but not limited to, endosomes andexosomes, respectively. The BSA/Dextran coated nanomagnetic particles soproduced typically have a PDI of 0.1. This PDI number is a measure ofthe width of the particle size distribution and is obtainedautomatically during DLS based size measurements. Generally,polydispersity indices less than 0.1 are typically referred to as“monodisperse” particle suspensions. More precisely, PDI=the square ofthe standard deviation divided by the mean diameter and is adimensionless number. Bioaffinity ligands, i.e., “targeting moieties”,such as antibodies and/or streptavidin, are then conjugated to the 110nm diameter BSA/Dextran coated nanomagnetic particles using standardhetero/homo-bifunctional coupling chemistries. Streptavidin-coatednanomagnetic particles so prepared are further heat-treated with a highionic strength salt solution (1 M to 5 M NaCl) in order to stabilize thesurface coatings on the particles.

In some embodiments, the targeting moieties associated with a targetednanomagnetic particle of the invention are labeled with a detectablelabel, for example, a radioisotope or fluorescent molecule, in order torender the particles, or the particle/targeted cell (or otherbiomolecular structure) complexes detectable through the use of acomplementary label detection instrument or system. Such labels can beincluded in the magnetic core particle and/or in one or more of theouter layers of a nanomagnetic particle of the invention. In otherembodiments where particle/cell detection is desired, a technology fordetecting the particle's magnetic signal may be employed, arepresentative example of which is SQUID technology, which can be usedto detect magnetic labels by virtue of the magnetic fields that theyproduce [Clarke and Braginski, SQUID Handbook, vol 1, (2004)].

In Vivo Applications

The targeted nanomagnetic particles of the invention can be adapted formany in vivo diagnostic and therapeutic uses, including imaging, celltherapies, and delivery of therapeutic agents to cells.

Cell Therapy

Today, many human diseases cannot be satisfactorily treated withstandard pharmaceuticals. For some of these diseases, cell therapiesoffer an attractive alternative. Cell-based therapies generally requiresignificant handling and processing of cellular products. Current celltherapy methods require substantial infrastructure and equipment to meetmanufacturing and regulatory requirements, including good manufacturingpractices, which involve the use of suitable clean rooms and personnelto maintain rooms, devices, production, quality control, and qualityassurance procedures under conditions that ensure non-contamination ofsamples to maintain sterility. Cell-based products are typicallyprocessed using a combination of different devices and disposables.Transfer of products and reagents in such processes can be manual and/orautomated.

Magnetic cell separation can include both enrichment and depletionprocedures. If target cells can be identified using cell surfaceproteins (or other cell surface biomolecules), they can be enriched tohigh purity through one or more rounds of enrichment and/or depletion.In other cases, target cells can be identified and removed from theresulting cellular product, which may be a heterogeneous mixture ofdifferent desired cells in which the number of cells targeted forremoval has been reduced. Of course, combinations of both enrichment anddepletion can be used.

Magnetic labeling of cells using targeted nanomagnetic particles of theinvention includes a suitable targeting moiety, typically a specificbinding member of a high affinity binding pair. The target cell/particlecomplexes can then be isolated using a magnetic separation device. Theisolation of multipotent cells, e.g., hematopoietic stem or progenitorcells, is of particular interest, although the present invention canapplied to a wide range of cell types or other biological materials orsamples.

Cellular products produced in accordance with the invention can be usedin therapy immediately or stored for later use using known methods.Formulation steps include adjusting the separated cell-containingpreparation to a desired volume or cell concentration, exchangingprocessing liquids with injectable solutions, adding stabilizers (e.g.,autologous plasma or serum, serum albumins, other proteins or syntheticpolymers, etc.) or adjuvants, supplementation with cryoprotective agentssuch as DMSO for subsequent storage, drawing of retention aliquots forquality control, delivery to combinations of bags or syringes forinfusion, etc.

Importantly, the targeted nanomagnetic particles of this invention canbe sterilized using any suitable method, including filter sterilized(due to the particles' small size) for use in therapeutic and/orin-vivo/in-vitro procedures where sterile processing is mandated ordesired.

In Vitro Applications

The targeted nanomagnetic particles of the invention can also be adaptedfor many in vitro diagnostic and therapeutic uses. Magnetic particleshave been used in the past to isolate or enrich eukaryotic cells,bacterial species, nucleic acids, and proteins. Beside particleisolation or cell separation, magnetic nanoparticles have also been usedto stimulate or activate cells by coating cell activating ligands on theparticle surface so that full three dimensional aspects of targetengagement, often important in biological systems, are more accuratelyreproduced as compared to solution phase activation protocols. In recentyears, magnetic particles have been studied for use in newer in-vitrotests. Examples of these include evaluation of the potential healtheffects of nanomagnetic particles (Kevin et al, Biosensors andBioelectronics, 43, 88 (2013)) and of nanotechnology-based systems fordelivery of si-RNA (Dim, et al, J. Nanobiotechnology, 13, 61 (2015)).Nanoparticles are also in research and development testing forapplication as targeted heating components that can develop localizedmagnetic hyperthermia conditions for the treatment of cancer (Makridis,et al., Mater Sci Eng C Mater Biol Appl., 63, 663 (2016).

EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in its practice.These Examples are in no way to be considered to limit the scope of theinvention in any manner, and those having ordinary or greater skill inthe applicable arts will readily appreciate that the specificationthoroughly describes the invention and can be readily applied to carryout the objects and obtain the ends and advantages mentioned, as well asthose inherent therein.

1. Synthesis of Silanized BSA/Dextran-Coated Nanomagnetic Particles

This example describes a preferred method for synthesizing silanizedBSA/Dextran coated nanomagnetic particles for use in the invention. Thissynthesis is carried out in three stages and involves first, thesynthesis of the bare (uncoated) magnetite superclusters followed by thesilanization of these superclusters, and, finally, a protein/polymercoating step using a mixture of BSA/Dextran.

Briefly, 5.02 g of ferrous sulfate and 7.22 g of ferric sulfate(SIGMA-ALDRICH; St. Louis, Mo.) are separately dissolved in 25 mL ofdegassed deionized water and then added into a reaction vesselcontaining 250 mL of degassed deionized water at 70° C. with continuousstirring. Next, 35 mL of 10 M ammonium hydroxide (SIGMA-ALDRICH; St.Louis, Mo.) is added into the reaction flask at a rate of 9 mL/minute,and the formation of the magnetite superclusters is allowed to proceedfor 30 minutes. The precipitate is then exhaustively washed withdeionized water using an in-house built ceramic low-gradient magneticseparator (LGMS) and finally stored under a nitrogen cap in degasseddeionized water. These magnetite superclusters typically have ahydrodynamic diameter in the range of 2 to 3 um as measured by dynamiclight scattering (Malvern Nano-S ZetaSizer; Westborough, Mass.).

Next, 1.65 g of the magnetite superclusters are sonicated in a 100 mLvolume of low ionic strength phosphate buffer (ACS grade monosodiumphosphate having a molecular weight of 137.99g/mole) with the aid of theVCX750 Ultrasonic processor (Sonics & Materials Inc., Newtown, Conn.)using a cooled, jacketed reaction beaker to a final size of ˜110 nm andthen immediately transferred into a reaction flask contained in asilicone oil-bath. Next, 2.5 mL of a 66 mg/mL of sodium silicate (SiO₂⁻)solution (SIGMA-ALDRICH; St. Louis, Mo.) is added into the reactionflask at a rate of 0.5 mL/minute followed by acidification with ˜8 mL of0.5M acetic acid added at 1 mL/minute to a final pH value of 6.0. Thetemperature of the oil-bath is then raised to 170° C. and the particlesuspension is allowed to dehydrate for about 3 hours in order to promotethe surface silanization of the nanomagnetic particles. After cooling toroom temperature, the particle suspension is placed into a LGMS magneticseparator for 30 minutes and the magnetically pelletized particles arerecovered and washed exhaustively with a low ionic strength HEPES buffer(VWR, Visalia, Calif.). These silanized or SiO₂-derivatized magnetiteclusters typically have a hydrodynamic size of about 200 nm and dissolvemuch more slowly in strong acid than their non-silanized versions (seeTable 1, below).

To prepare the BSA/Dextran coated nanomagnetic particles, 1.4 g of thesilanized magnetite clusters are sonicated in a 135 mL volume of lowionic strength phosphate buffer with the aid of the VCX750 Ultrasonicprocessor (Sonics & Materials Inc.; Newtown, Conn.) using a cooled,jacketed reaction beaker to a final size of ˜100 nm and then immediatelytransferred into a 1 L jacketed reaction flask thermostated to 70° C.that contains 400 mL of a mixture of 20 mg/mL BSA (Lampire Biologicals;Pipersville, Pa.) and 0.2 mg/mL Dextran (SIGMA-ALDRICH; St. Louis, Mo.).This coating reaction is allowed to proceed for 30 minutes at 70° C. Thecoated nanomagnetic particles are then cooled to room temperature andleft undisturbed overnight at 4° C.

Next, the particles are slowly decanted from the vessel with the aid ofa low-field ceramic magnet held at the bottom of the vessel in order tosediment away the large size (˜300 nm) particle aggregates. Thecollected supernatant (of ˜100 nm diameter) is then subjected to 7cycles of high-field magnetic washes in low-ionic strength HEPES buffer(VWR; Visalia, Calif.). These high-field washes, in addition to removingthe excess reactants, also serves to significantly narrow the particlesize distribution to values of ≤0.1 PDI. The final hydrodynamic particlesize is typically about 115 nm. The overall yield starting from 1.65 gof the magnetite superclusters is typically at least 50%. The firsthigh-field magnetic wash supernatant, which typically has a hydrodynamicsize of ˜70 nm and which constitutes ˜35% of the total particle yield,is collected as a by-product and can be used to produce smaller size(<100 nm) nanomagnetic particle products for use as an in-vivo/in-vitrotracking/capture label as well as for magnetic cell isolations inconcert with HGMS columns (see EXAMPLE 3, below).

Finally, a member of a bioaffinity ligand pair such as Streptavidin,antibodies, or other desirable ligands can be covalently conjugated tothe ample BSA-derived functional groups present on these BSA/Dextrancoated nanomagnetic particles using standard hetero/homo-bifunctionalconjugation chemistries as will be familiar to those skilled in the art.

2. Synthesis of Nanomagnetic particles by Peptization and Other Types ofSilanizing Agents

Electrolytes such as the chelating agents known more popularly as EDTA,EGTA, as well as weak bases and acids are referred to as peptizingagents in instances where they help to disperse precipitates intocolloidal sols. In this example, EGTA (SIGMA-ALDRICH; St. Louis, Mo.),which is a strong iron chelating agent, is added (at 0.25 molesEGTA/mole iron) immediately after the formation of the magnetitesuperclusters as in Example 1, above. This chelation step is allowed toproceed for 1 hour at 70° C. prior to washing up the magnetitesuperclusters as in Example 1, above. Unlike the ˜2.5 um size of thestarting magnetic superclusters, these EGTA peptized magnetite clusterstypically have a hydrodynamic diameter of about 1 um, and such a sizereduction is indicative of a successful dispersion of the magnetitesuperclusters.

In another embodiment, a sequential silanization method is used wherebyEGTA peptized magnetite superclusters are sonicated and silanized as inExample 1, above, and immediately after the addition of the sodiumsilicate solution, an equimolar amount of the amino-functionalizedsilanizing agent aminopropyltrimethoxysilane or APS (SIGMA-ALDRICH; St.Louis, Mo.) is added and the particles allowed to dehydrate for 90minutes at 160° c after acidification to pH 6.0. Silanization has alsobeen achieved using just APS in lieu of sodium silicate as in Example 1,above, and the dehydration step allowed to proceed for 75 minutes at160° C.

Another silanization agent, hydroxymethyltriethoxysilane (Gelest,Morrisville, Pa.), is very hydrophilic, and has also been successfullyused to produce silanized nanomagnetic particles useful in the contextof the invention. In this particular case, 15 wt % of this silanizingagent (relative to the iron content) was used and the dehydration wasallowed to proceed for 2 hours at 160° C.

All of the aforementioned silanized magnetic particles have beensuccessfully coated with BSA/Dextran mixtures as described in Example 1,above. These types of silanized nanomagnetic particles, whenencapsulated with BSA/Dextran mixtures, typically exhibit 50% aciddissolution after 15 minutes exposure to 4 M HCL. Briefly, to performdissolution, 100 uL of 0.1 mg/mL (in terms of iron content) of theparticle suspension was incubated with 200 uL of 6 M hydrochloric acidand aliquots of this mixture were removed at various time intervals andassayed for the presence of elemental (or dissolved) iron bycomplexation with potassium thiocyanate as a colorimetric endpointreadout. Table 1, below, shows the acid dissolution behavior of all ofthese aforementioned silanized magnetite clusters.

TABLE 1 Percent dissolution of Iron oxide as a function of acid exposuretime for various silanized magnetite clusters Time in 4M APS + SilicateAPS only Hydroxymethyl- Hydrochloric Silanized silanized EGTA silanizedsilanized acid Magnetite magnetite peptized magnetite magnetitemagnetite (minutes) Superclusters clusters clusters clusters clusters 543.8% 22.8% 21.8% 33.1% 28.1% 10 72.5% 33.7% 38.8% 47.1% 49.1% 15 90.0%48.4% 55.0% 64.9% 68.9% 30  100% 77.0%  100% 89.5%  100% 45  100%  100% 100%  100%  100%

The data in Table 1, above, show that the silanization methods describedherein indeed provide protection against acid dissolution and also serveto provide highly cross-linked silane molecules on the surface of themagnetic particles. For instance, at the 15 minute time point, 90% ofthe (bare) magnetite superclusters were dissolved by acid compared toonly 50% to 70% of the silanized magnetite clusters.

FIG. 1 shows the results of an acid dissolution study performed on asilanized nanomagnetic particle pre- and post-sonication. This studyshows that the silane (glass) coating remained intact on thenanomagnetic particle surface after the second round of high powersonication as described in Example 1, above.

Nanomagnetic particles produced without a primary glass coating aretypically not stable in biological fluids such as plasma and wholeblood, and, furthermore, they are prone to aggregation even in solutionsof low ionic strength. Such protective functionalities (e.g., stability,reduced aggregation) provided by the silanization processes describedherein significantly contribute to the practical utility of the targetednanomagnetic particles claimed in this patent in biological researchefforts as compared to other magnetic nanoparticles.

3. Derivitization, Processing and Cell Labeling Efficacy of the 70 nmBSA/Dextran-Coated Silanized Nanomagnetic Particle By-Products

The first high-field magnetic wash supernatant from Example 1, above,the magnetic particles in which had a hydrodynamic size of about 70 nm,was first subjected to HGMS purification using a commercially availableHGMS ‘XS’ column (Catalogue# 130-041-202; Miltenyi Biotec, San Diego,Calif.), which is packed with small ferromagnetic beads in order toremove the excess coating reagents. The ‘XS’ column was positioned in auniform magnetic field created by positioning a 2 inch×1 inch×0.25 inchthick ‘North’ face and an identically sized ‘South’ face magnet againsteach other. The ‘XS’ column/magnetic assembly was attached to aperistaltic pump to facilitate rapid automated processing of thenanomagnetic particles.

30 mL (12.5 mg iron) of the first high-field magnetic wash supernatantwas HGMS purified into a low-ionic strength HEPES pH7.5 buffer. Afterremoval of the ‘XS’ column from the uniform magnetic field andresuspension with 3 mL of HEPES pH7.5 buffer, about 90% of the particleswere recovered based on iron content. These HGMS-purified nanomagneticparticles had a hydrodynamic diameter of 73 nm and were then conjugatedto a rat anti-mouse CD4 antibody (Catalogue# 100506; BioLegend Inc., SanDiego, Calif.) using heterobifunctional coupling chemistry. Briefly, theHGMS-purified 73 nm BSA/Dextran-coated nanomagnetic particles wereactivated with a SMCC cross-linker (Catalogue# 51534; ThermoFisherScientific, San Diego, Calif.) and conjugated to the rat anti-mouse CD4antibody which had been thiolated using 2-Iminothiolane (Catalogue#26101; ThermoFisher Scientific, San Diego, Calif.). The finalhydrodynamic size of these antibody-conjugated nanomagnetic particleswas measured to be 83 nm. Although not thoroughly optimized, when thisconjugated particle was used for targeting mouse CD4⁺ cells fromsplenocyte cell suspensions in conjunction with ‘MS’-type HGMS columns(Catalogue# 130-042-201; Miltenyi Biotec Inc., Auburn, Calif.), puritiesand yields in excess of 90% were obtained as measured by flow cytometrywith appropriate fluorescently labeled antibodies (FACSCalibur withCellQuest software; BD Biosciences, San Diego, Calif.).

These results show that these smaller particles, as compared to thelarger ones described elsewhere herein, can also be effectivelyconjugated and utilized for isolation of biological materials.

4. Colloidal Stability of Streptavidin-Conjugated Nanomagnetic Particles

A 115 nm diameter BSA/Dextran-coated nanomagnetic particle producedaccording to Example 1, above, was conjugated covalently to Streptavidinas per the methods described in Example 3, above, to produce 135 nmdiameter Streptavidin-conjugated nanomagnetic particles. Particle sizemeasurements were carried out at various time points after resuspendingand storing the nanoparticles in a high ionic strength solution (1 MNaCl) at room temperature. Control size measurements were carried out onthe same nanoparticles in their normal storage buffer, which was alow-ionic strength buffer supplemented with BSA and sodium azide. Table2, below, shows the results of this study. This study demonstratessignificant resistance to aggregation and enhanced colloidal stabilityof the nanomagnetic particles of this invention.

TABLE 2 Particle Size Stability in 1M Sodium Chloride STORAGE SIZE @SIZE @ SIZE @ SOLUTION 0 HOURS 1 HOUR 72 HOURS Normal Storage Buffer 135nm 137 nm 137 nm 1M Sodium Chloride 139 nm 139 nm 140 nm

5. Magnetic Separation Efficiency of Nanomagnetic Particles of theInvention

FIG. 2 shows the magnetic separation efficiency of various nanomagneticparticles made according to this invention, which particles include a113 nm diameter BSA/Dextran coated, a 127 nm antibody-conjugatedparticle, and a 130 nm Streptavidin-conjugated particle, the latter twoof which are conjugated using the method described in Example 3, above.This study was performed using quadrupole magnetic separator built asdescribed in U.S. Pat. No. 5,186,827 and designed to fit standard 12mm×75 mm disposable laboratory test-tubes with dilute nanomagneticparticle suspensions containing 25 ug/mL iron in a physiological buffersuch as an isotonic phosphate buffered saline solution. Similar strongmagnetic separators for use with test-tubes are available from StemCellTechnologies (Part #18000; Vancouver, British Columbia, Canada). FIG. 2shows that all these aforementioned nanomagnetic particles separaterapidly and quantitatively within just a few minutes.

Antibody-conjugated commercially available microbeads (Catalogue#130-049-201; Miltenyi Biotec Inc., Auburn, Calif.) having a measuredhydrodynamic diameter of 82 nm were also tested for magnetic separationefficiency in the quadrupole magnetic separator, and the results shownin FIG. 3. As shown in FIG. 3, these 82 nm microbeads did notquantitatively separate at all in the quadrupole magnetic separatordescribed above in this example. Instead, only about 40% of thosemagnetic particles could be separated after 30 minutes. These resultsdemonstrate that those types of microbeads are only suitable for usewith HGMS column-based magnetic separation methods. The nanomagneticparticles of the present invention, however, are suitable forquantitative magnetic particle-based separations in both external-field(dipole, tripole, quadrupole, hexapole type) as well as internal-field(HGMS)-based magnetic separators. This property represents a significantdifferentiator in terms of practical utility of the nanomagneticparticles described in this specification, as no other magneticparticles are presently known to the inventors to function in bothinternal and external types of magnetic separators.

6. Non-Specific Binding of Nanomagnetic Particles to Mammalian Cells

Table 3, below, shows the non-specific binding (NSB) of different lotsof BSA/Dextran-coated nanomagnetic particles synthesized over a 6 monthperiod according to Example 1, above. In this study, mouse splenocytes(1×10⁷ total cells per tube) were incubated for 20 minutes at 4° C. witha relatively large number of nanomagnetic particles (about 2000particles/cell or 2×10′° total number of particles per sample). Thecell/nanomagnetic particle reaction mixture was then magnetically washedtwice with the aid of a quadrupole magnetic separator using just 5minute magnetic separation times.

The washing steps were performed as follows: the cell suspension wasdiluted to a total volume of 4 mL with an isotonic PBS/BSA/EDTA buffer(5× Phosphate buffered saline (PBS), pH 7.2, 2.5% (w/v) Bovine SerumAlbumin (BSA), and 10 mM EDTA) and the tube placed into aquadrupole-magnetic separator for 5 minutes. The supernatant was thendiscarded by gentle inversion of the magnetic separator or by aspirationwith the aid of a pasteur pipette. The tube was then removed and itscontents resuspended again with 4 mL of the isotonic buffer and placedback into the magnetic separator for another 5 minute separation. Afterthe second aspiration, the cells were centrifuged and the cell pelletwas resuspended with a small volume of isotonic buffer and then analyzedfor the presence of non-specifically collected cells. The magneticallycollected cells were then centrifuged once (5 minutes at 300×g) toremove excess or free nanomagnetic particles. The number of magneticallycollected cells was then counted using an automated cell counter(Cellometer® VISION Trio; Nexcelom Bioscience, Lawrence, Mass.) which,together with the starting number of cells, enabled calculation of thepercentage of cells that were magnetically selected (which is referredto as non-specific binding). Note that 2×10′ particles is equivalent toa mass of about 40 ug of iron. More typically, for the efficientisolation of cells in high purity and yield, only about 10 ug to 20 ugof nanomagnetic particles (in terms of iron weight) need to be added fora sample containing 1×10⁷ total cells.

These non-specific binding experiments were also repeated using highgradient magnetic separation (HGMS) columns (Part #130-042-201;MS-Columns; Miltenyi Biotec Inc., Auburn, Calif.) in place of thetest-tube quadrupole magnetic separator (see Table 3, below). Due to thevery high magnetic field gradients generated in such HGMS columnseparators, the nanomagnetic particle-to-cell ratio was drasticallyreduced to about 20 particles per 1 cell, or about 2×10⁸ particles persample. It was discovered that particle-to-cell ratios from 10:1 to 50:1provide optimal target cell yields and purities (see Tables 5 and 6,below).

These studies were performed with a commonly used standard, cellcompatible buffer (PBS) supplemented with 0.5 wt % BSA, 2 mM EDTA, and0.1 wt % Casein and adjusted to pH 7.2.

TABLE 3 Particle Particle Magnetic % Non-Specific Lot # Diameter (nm)Separation Method Binding MAG05 110 Quadrupole 1.1 MAG06 113 Quadrupole1.5 MAG07 110 Quadrupole 1.9 MAG08 105 Quadrupole 1.1 MAG09 114Quadrupole 1.4 MAG10 114 Quadrupole 1.1 MAG05 110 MS Column 1.1 MAG06113 MS Column 1.0 MAG07 110 MS Column 1.0 MAG08 105 MS Column 1.2 MAG09114 MS Column 0.8 MAG10 114 MS Column 1.1

The non-specific binding (NSB) results in Table 3, above, with thenanomagnetic particles of this invention are extremely low, making itpossible to attain target cell purities of up to about 99%. Most, if notall of currently available commercial magnetic particles cannot attainsuch low levels of NSB.

7. Specific Binding and Capture of Mammalian Cells Using a QuadrupoleMagnetic Separator Compared to HGMS Separators

Table 4, below, shows the titration results of a 127 nm diameter ratanti-mouse CD4 antibody—(Clone RM4-5; catalogue#100506; BioLegend Inc.,San Diego, Calif.) conjugated nanomagnetic particle (prepared asdescribed in Example 3, above) with mouse splenocytes. This titrationwas performed using particle-to-cell ratios from 500:1 to 1500:1. Theprotocol used was essentially identical to that described above inExample 6, above, except that after removal of excess nanomagneticparticles, an additional incubation with appropriatefluorochrome-conjugated antibodies (for phenotyping purposes) wascarried out and the cells analyzed on a flow cytometer (FACSCalibur withCellQuest software; BD Biosciences, San Diego, Calif.) to determine thepercent purity of the magnetically selected cells.

TABLE 4 Particle-to-Cell Ratio % Purity % Yield  500:1 93.0 91.2  750:191.6 90.2 1000:1 92.6 92.9 1200:1 92.1 95.1 1500:1 89.3 90.7This study clearly illustrates the biomedical utility of thenanomagnetic particles of this invention for isolating target cells ofinterest in high yield and purity for further interrogation.

Table 5, below, shows the results of a similar titration study doneusing the same 127 nm diameter rat anti-mouse CD4 antibody-conjugatednanomagnetic particle with mouse splenocytes; however, in this study,HGMS columns were used for performing the magnetic wash steps. Asdescribed to earlier (in Example 6, above), lower particle-to-cellratios, from 5:1 to 50:1, were used in this HGMS based study.

Table 6, below, shows the results of a similar titration study done alsousing HGMS columns but with a 129 nm diameter rat anti-mouse CD19antibody- (Clone 6D5; catalogue#115502; BioLegend Inc., San Diego,CA92121) conjugated nanomagnetic particle (prepared as described inExample 3, above) in order to demonstrate the versatility of thenanomagnetic particles of the invention in HGMS-based cell isolationprotocols.

TABLE 5 Particle-to-Cell Ratio (rat anti-mouse CD4) % Purity % Yield 5:1 90.1 55.9 10:1 92.5 84.9 20:1 88.9 95.8 30:1 84.5 98.6 40:1 84.299.1 50:1 83.0 99.2

Particle-to-Cell Ratio (rat anti-mouse CD19) % Purity % Yield 10:1 98.182.1 20:1 97.8 96.5 30:1 97.4 98.0 40:1 96.7 98.4 50:1 96.6 97.9

Both of these studies yielded excellent results for the purity and yieldof the magnetically (purified) captured target cells across a relativelywide range of particle to cell ratios.

A commercially available magnetic particle was measured to have ahydrodynamic diameter of 170 nm and was also titrated as described inthis example, with the results being shown in Table 7, below.

TABLE 7 Particle-to-Cell Ratio % Purity % Yield 10:1 93.7 88.7 15:1 91.060.0 20:1 95.2 62.0 30:1 93.9 37.0

This commercially available magnetic particle did not exhibit asufficiently wide particle-to-cell usage ratio such that reliable andreproducible results could be obtained, therefore indicating that suchconventional magnetic particles are not compatible for use withHGMS-type magnetic separation methods. The rapid loss of yield upontitration with those magnetic particles was most likely due toentrapment of the relatively large sized magnetic particles in themetallic (or ferromagnetic) matrix in the HGMS column, leading toinefficient recoveries of the magnetically labeled cells retained on thede-magnetized HGMS column. As those in the art will appreciate, suchconventional magnetic particles can only be practically used with strongexternal-field magnetic separators such as the quadrupole-type magneticseparators used in the studies described above.

8. Stability of the Nanomagnetic Particles Produced According toExamples 1 and 3, Above

To assess the long-term stability of the nanomagnetic particles of thisinvention, both Streptavidin- and rat anti-mouse CD19antibody-conjugated particles were prepared according to Examples 1 and3, above. Multiple small aliquots of these particles were then stored atthree different temperatures (4° C., 25° C., and 37° C.) and magneticcell separation tests were performed at various time points over thecourse of two months in order to monitor the overall biostability ofthese nanomagnetic particles. BioLegend's MojoSort™ Mouse CD4 T CellIsolation Kit (Catalogue# 480005) is a negative selection test that usesStreptavidin nanomagnetic particles in conjunction with a cocktail ofbiotinylated antibodies in order to magnetically select all cells thatare CD4 negative. Additionally, BioLegend's MojoSort™ Buffer andMojoSort™ Magnet were used in the execution of the cell selectionprotocols described in this example. The ‘“untouched” cells orsupernatant from the magnetic separation step contained the desiredCD4-positive cells. These “untouched” cells were then analyzed on a flowcytometer (FACSCalibur with CellQuest software; BD Biosciences, SanDiego, Calif.) in order to determine the purity and yield of thetargeted CD4-positive cells. Similar analyses were also performed usingBioLegend's MojoSort™ Mouse CD19 Nanobeads (BioLegend, Catalogue#480001), which are rat anti-mouse CD19 antibody-conjugated nanomagneticparticles used to positively select CD19-positive cells.

FIGS. 4 and 5 show the purity and yield, respectively, of CD4 positivecells that were negatively selected using Streptavidin nanomagneticparticles and appropriate biotinylated antibodies that were stored atvarious temperatures and tested for cell separation performance over thecourse of two months.

Similarly, FIGS. 6 and 7 show the purity and yield, respectively, of ratanti-mouse CD19 antibody conjugated nanomagnetic particles that werestored at various temperatures and tested for cell separationperformance over the course of two months. Note that a noncross-reacting, fluorescently labeled B-cell-specific antibody calledCD45R/B220 (Catalogue# 103223; BioLegend Inc., San Diego, Calif.) wasused to identify the magnetically selected B cells.

The results of these stability studies clearly demonstrates theexcellent biostability of the nanomagnetic particles of the invention.The fact that both sets of nanomagnetic particles used in these studieshave at least 30 or more days of biostability at an elevated temperatureof 37° C., which can be extrapolated to upwards of more than 4 years ofbiostability at 4° C., can be attributed to the patentable nanomagneticparticle composition and synthesis designs presented in thisspecification. In contrast, conventional magnetic particles ranging insize from 80 nm to 1000 nm have been reported to have shelf-lives orbiostability in the range of a few months to about 20 months even whenrefrigerated at 4° C.

9. Comparison of Particle Size Distributions

The particle size distributions of various conventional, commerciallyavailable magnetic particles that are highly utilized in the targetedcell separations market were measured and compared to those producedusing Example 1, above. These measurements were made using dynamic lightscattering (Malvern Nano-S ZetaSizer; Westborough, Mass.), and thepercentage of particles in various ‘size-bins’ was plotted as a functionof actual particle size, as shown in FIG. 8. Hydrodynamic diameters aremeasured on a Malvern Nano-S ZetaSizer instrument that uses theprinciples of ‘dynamic light scattering’ whereby particles areilluminated with a laser and the scattered light analyzed for intensityfluctuations. The nanomagnetic particles of the invention (labeled as“BioLegend” in FIG. 8) had a hydrodynamic diameter of about 130 nm andrelatively insignificant numbers of particles greater than about 300 nmin diameter (an important criterion in order for magnetic particles toperform equally well in both ‘external-field’ and ‘internal-field’ basedmagnetic separators). Note that the particles labeled “ConventionalParticle ‘A’” in FIG. 8 had a hydrodynamic diameter of about 82nm andtherefore would only be suitable for use with ‘internal-field’generating or HGMS columns (see FIG. 3, above, also).

10. Transmission Electron Microscopy of Cells Selected Using HGMS

In this study, cells were magnetically selected by HGMS using bothcommercially HGMS compatible magnetic particles (FIG. 9, Panel A) andtargeted nanomagnetic particles of the present invention (FIG. 9, PanelB). The representative electron micrographs shown in FIG. 9 wereproduced using 55 nm cryosections of the magnetically selected cells andimaged on a Transmission Electron Microscope. A single cell suspensionfrom C57BL/6 mouse spleen was prepared to isolate CD19+ B cells usingthe MojoSort™ Mouse CD19 Nanobeads (BioLegend, Calif.) and commercialmouse CD19 MicroBeads (Miltenyi, Germany) followed by BioLegend andMiltenyi recommended protocol. Isolated CD19 cell purity (97% fromBioLegend, 94.9% from Miltenyi) was identified by staining of theresulting cells with CD45R/B220 (clone RA3-6B2) PE and analysis by flowcytometry. Then the cells were centrifuged and the cell pellets wereresuspended in a modified Karnovsky's fixative (2.5% glutaraldehyde and2% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) for 4hours. Then the preparation was post-fixed in 1% osmium tetroxide in0.15 M cacodylate buffer for 1 hour and stained en bloc in 2% uranylacetate for 1 hour. Samples were then dehydrated in ethanol, embedded inDurcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on aLeica UCT ultramicrotome, and picked up on formvar and carbon-coatedcopper grids. Sections were stained with 2% uranyl acetate for 5 minutesand Sato's lead stain for 1 minute. Grids were then viewed on a JEOL1200EX II (JEOL, Peabody, Mass.) transmission electron microscope andphotographed using a Gatan digital camera (Gatan, Pleasanton, Calif.),or viewed using a Tecnai G2 Spirit BioTWIN transmission electronmicroscope equipped with an Eagle 4k HS digital camera (FEI, Hilsboro,Oreg.).

Similarly low numbers of the targeted nanomagnetic particles of theinvention compared to those of conventional labeled magnetic particleswere observed across 40 images from each sample type. These electronmicrographs clearly show that far fewer of targeted nanomagneticparticles of the invention are bound to the target cells than in themicrographs showing cells bound by conventional labeled magneticparticles. The arrows in these micrographs mark the location ofvisualizable magnetic particles on the surface of these cells. This (theability to mediate magnetic separation with very few nanomagneticparticles per cell) is a very important attribute of the targetednanomagnetic particles of the present invention because suchmagnetically selected cells are essentially in a “native” or “untouched”state with very little, if any, perturbation of the cell's biologicalprocesses. This allows the cells to be captured in a biologically intactand responsive state (see Example 12).

11. Nanomagnetic Particle Lyophilization Studies

Mouse anti-CD19 conjugated nanobeads (2×10⁸ total particles) and SAvconjugated nanobeads (2×10⁸total particles) produced according toExamples 3 and 4, above, respectively, were suspended in varioussupplemented solutions and subjected to a 3 day lyophilization (Lyo)cycle on a Genesis Pilot Lyophilizer (SP Scientific). Specifically,particle suspensions contained in silanized glass vials were frozen downto −46° C., then to −80° C. for 3 hours and back to −46° C. and kept ina sealed vacuum chamber for 3 days. Thereafter, the temperature wasraised to 22° C. The lyophilized nanomagnetic particles were thenreconstituted with PBS and tested for performance using both theMojoSort™ Mouse CD19 Nanobeads (BioLegend Inc., San Diego, Calif.;catalogue #480001) and the MojoSort™ Mouse CD4 T Cell Isolation Kits(BioLegend Inc., San Diego, CA; catalogue #480005). The results shown inTables 8 and Table 9, respectively, below.

TABLE 8 Mouse CD19 positive selection purity and yield by usingreconstituted lyophilized (lyo) CD19 nanobeads Particles Purity (%)Yield (%) Non Lyophilized 6D5 particle (Control) 97.7 82 6D5 nanobeadsin Storage Buffer (Lyo) 97.1 72 in 1% BSA (Lyo) 96.9 88 in 1% Dextran(Lyo) 96.9 88.2 in 2% Sucrose (Lyo) 97 89.2 in 1% Dextran + 1% Sucrose(Lyo) 96.7 90.8

TABLE 9 Mouse CD4 negative selection purity and yield by usingreconstituted lyophilized (lyo) SAv particles Particles Purity (%) Yield(%) Non lyophilized SAv (Control) 95.4 90.0 in 1% BSA (Lyo) 92.8 92.9 in1% Dextran (Lyo) 96 88.1 in 2% Sucrose (Lyo) 96 87.0 in 1% Dextran + 1%Sucrose (Lyo) 96.2 87.6

These lyophilized and reconstituted nanomagnetic beads show excellentretention of bioactivity, indicating that lyophilization facilitatesextended storage/stability of the targeted nanomagnetic beads of theinvention for very long periods of time.

12. Functional Studies of Magnetically Selected Cells

Magnetically selected cells are often used for downstream processingsuch as gene/protein/RNA profiling; however, many if not most ofcommercially available magnetic particles have a toxic effect on cells,Therefore, it is quite challenging to obtain live or viable cells withmagnetic particles attached to them for further studies/probing. In thisstudy, both a targeted nanomagnetic particle of the invention and awidely used commercially available magnetic particle conjugated to anantibody against the mouse CD4 antigen were tested side-by-side for cellfunctionality after the target CD4+ cells were magnetically isolated.

Briefly, a rat anti-mouse CD4 antibody (Clone RM4-5; catalogue#100506;BioLegend Inc., San Diego, Calif.) conjugated nanomagnetic particle(prepared as described in Example 3, above) was tested alongsideanti-CD4 (Clone L3T4; Catalogue# 130-049-201; Miltenyi Biotec Inc.,Auburn, Calif.) conjugated microbeads using HGMS columns (Catalogue#130-042-201; Miltenyi Biotec Inc., Auburn, Calif.). Theanti-CD4-conjugated nanomagnetic particles of the invention had ahydrodynamic diameter of 127 nm whereas the L3T4-conjugated microbeadshad a hydrodynamic diameter of 82 nm. Table 10, below, shows the purityand yield of the isolated CD4+ cells from both types of these magneticparticles when used for isolating CD4+ cells from a mouse spleenaccording to the manufacturer's instructions.

TABLE 10 Type of nanomagnetic particle used % PURITY % YIELD BioLegendanti-CD4 nanobeads 92.4 65 MACS anti-CD4 MicroBeads 91.5 67

After magnetic isolation of the CD4+ cells, equal amounts of CD4+ cells(1×10⁶ cells) from both isolation methods were seeded into 96-wellmicroplates coated with mouse anti-CD3 (Clone 17A2; catalogue# 100201;BioLegend Inc., San Diego, Calif.) antibody in varying concentrationsand supplemented with 1 ug/mL soluble mouse anti-CD28 (Clone 37.51;catalogue#102101; BioLegend Inc., San Diego, Calif.) and incubated for 3days at 37° C. Next, a solution of the fluorescent redox markerresazurin (catalogue# TOX8-1KT; SIGMA-ALDRICH; St. Louis, Mo.), whichmeasures the metabolic activity of living cells, was added into thewells at a 10% volume ratio and the relative fluorescence intensity wasmeasured after a 7 hour incubation using a SPECTRAmax Gemini XPSfluorescence microplate reader (Molecular Devices, Sunnyvale, Calif.). Aplot of the relative fluorescence units (RFU) versus the concentrationof the anti-mouse CD3 antibody used for coating the microwells is shownin FIG. 10. Note that in FIG. 10, the higher the fluorescence intensity,the higher the number of living cells.

The results of this functional cell assay clearly demonstrates that thenanomagnetic particles of the present invention do not have asignificant toxicological effect on the magnetically selected cells eventhough these nanomagnetic particles are larger than the testedcommercial magnetic microbeads.

13. Combined Use of Microbubbles in Conjunction with NanomagneticParticles for Cell Isolation

Micro- sized buoyant bubbles are hollow (or air-filled) micron-sizedspheres that are commercially available with functionalized surfaces orcoated ligands for targeting moieties of interest. Commerciallyavailable examples that could be conjugated with cell-specific ligands(e.g., cell antigen specific antibodies) and used to isolate specificcell populations include the gas-encapsulated microbubbles from Targeson(San Diego, Calif.) and Buoyant Microbubbles from Akadeum Life Sciences(Ann Arbor, Mich.). Examples described in the research literatureinclude the perfluorocarbon microbubbles of Shi, et al., Methods, 64,102 (2013), glass microbubbles of Hsu, et al., Technology (SingaporeWorld Science), 3, 38 (2014), albumin microbubbles of Liou, et al., PLoSOne, 20, 10 (2015), and gas-filled immune-microbubbles of Shi, et al.,PLoS One, 8, 1 (2013). Examples of patent literature describing the useof microbubble-based systems for isolation of analytes or cells includeU.S. patent and published patent application U.S. Pat. Nos. 5,116,724,5,246,829, 8,835,186, US 2003/0104359 A1, US 2007-0036722 A1, and US2011/0236884 A1. These examples illustrate the value of using abuoyancy-based system for the specific isolation of target cells andanalytes. Yet, prior to this invention, none have combined a buoyancybased system with magnetic nanoparticles to provide faster, moreefficient and more effective isolation of the desired target(s).

In this example, a patentable method is described wherein both targetedmicrobubbles, of any composition, and targeted nanomagnetic particles,of any composition, can be used sequentially and/or simultaneously toobtain one, two, or three cell populations of interest. A combination ofmagnetic and buoyant isolation, or “magnetibuoyant”, procedures willallow difficult separations to be achieved. Such “magnetibuoyant”methods of cell isolation significantly reduce the time and resourcesrequired to isolate different cells of interest, and the populations canbe obtained at very high purities. The magnetic nanoparticles of thepresent invention are particularly well suited for this application dueto their high stability in various fluids, small size, more highlymagnetically responsive property, ability to separate cells at lowerparticle to cell ratios as compared to other magnetic particles andcapacity to respond quickly to magnetic fields as compared to othermagnetic particles. These advantages have not previously been realizedand/or commercialized. FIG. 11 illustrates the general principle.

Considering FIG. 11, if a mixture of different cell types (A, B, C, D)containing two desired subpopulations (A and B) are combined in areaction mixture with microbubbles targeted to one cell type (A) andwith magnetic particles targeted to a second type (B), then allowing thefirst set of A cells to float to the surface while the second set of Bcells is drawn to a strong magnetic field (such as the quadrupolemagnetic separator described in Example 5, above), this will cause themagnetized target cells to be separated at right angles to thelevitation direction of the microbubble-targeted cells. In this mannerboth populations of cells can be isolated at the same time and can beharvested individually for further use from the same initial reactionmixture. In this simple example both of the different cell populations(A and B) may be desired for further use, and can be easily harvested.Alternatively, one population may be unwanted cells that will bediscarded with, for example, the intent of removing them as potentialcontaminants of the second isolated population. And finally, the third“remainder” population (in this example, cell types C and D), i.e.,those not targeted by either the microbubbles or the magnetic particles,may also be harvested for further use since that(those) population(s)can also be retained as the two targeted populations (A and B) areharvested.

As background, FIG. 12 illustrates the principle of using onlymicrobubbles to isolate mouse CD19+ cells. In this example, ratanti-mouse CD19 conjugated (Clone 6D5; catalogue#115502; BioLegend Inc.,San Diego, Calif.) microbubbles (prepared using the conjugation methodsdescribed in Example 3, above, except that centrifugation is used forall wash steps) are incubated with mouse splenocytes for 15 minutes at4° C. in a small eppendorf tube on a rotator (see (a) in FIG. 12). Thecell suspension is then transferred into a test tube and diluted up to atotal volume of 4 mL with isotonic cell buffer and centrifuged for 5minutes at 300×g (see (b), FIG. 12). The floating cells are then gentlypoured or aspirated and transferred into a new test-tube (see (c), FIG.12). The cells are then stained with a fluorescent CD45R/B220 antibodyconjugate (phycoerythrin conjugated rat anti-mouse/human CD45R/B220;catalogue# 103207; BioLegend Inc., San Diego, Calif.), and the collectedcells analyzed on a flow cytometer (FACSCalibur with CellQuest software;BD Biosciences, San Diego, Calif.). T able 11, below, shows the purityand yield of the mouse CD19+ target cells obtained using such antibodyconjugated microbubbles.

TABLE 11 Pre Isolation Post Isolation Purity  55% 98.6% Yield 100% 92.4%

Example i: Magnetibuoyant Methods for Rare Cell Isolation

Commercially available methods for isolating rare cells (i.e., cellssuch as stem cells, circulating tumor cells, fetal cells, endothelialcells, etc.) are magnetic particle-based, two-step protocols where anegative depletion step is carried out first to remove unwanted cellsfollowed by washing steps and a positive selection step to capture rarecells. The direct positive selection of rare cells has only limitedsuccess due to non-specific binding of the solid-phase materials (i.e.,magnetic and non-magnetic beads) and the immense difficulty in targetingand binding to these rare cells, which are present only at very lowfrequencies, typically at 1 target cell per 1 million (or more) totalcells. Furthermore, the starting cell suspensions often used for directpositive selection of rare cells are very challenging cell preparationssuch as whole blood, buffy coats, and/or lysed whole blood. Anysignificant manipulation of the starting or native cell suspension has anegative impact on the recovery/yield of any rare cells present in thesample due to inherent cell losses experienced at every stage of cellsample processing.

FIG. 13 depicts a protocol for using CD45 antibody- (Clone2D1-anti-human CD45; catalogue #368502; BioLegend Inc., San Diego, CA)conjugated microbubbles. The CD45 microbubbles are used in conjunctionwith a rare cell-specific, CD326 antibody (Clone 9C4-anti-human CD326[EpCAM]; catalogue #324202; BioLegend Inc., San Diego, Calif.) that isconjugated to a nanomagnetic particle (preferably, a nanomagneticparticle prepared in accordance with this invention). Used together, thecombination of particles allows the direct and efficient enrichment ofcirculating tumor cells in a single step.

In FIG. 13, (a) represents the reaction mixture in a standard tissueculture tube compatible with a commercially available magneticseparator. After an incubation step to allow the targeted buoyant andmagnetic particle populations to bind to their respective target cells,the tube is inserted into a magnetic separator where the magneticparticles and cognate cells are drawn to the magnetic field on the wallsof the tube, At the same time, the microbubbles levitate their cognatecells to the surface (b). With the tube still in the magnetic separator,the microbubble-associated cells can be aspirated or simply poured awaywithout disturbing the magnetically retained cells. After decanting thetube (b) and removing it from the magnetic separator, a very puresuspension of these rare tumor cells is left behind in the tube (c) forfurther interrogation and studies.

Example ii: Magnetibuoyant method for isolating human CD4+ cells at veryhigh purities

The rationale depicted in FIG. 13 above can also be applied to theisolation of human CD4+ lymphocytes since the antibody targeting the CD4antigen receptor is also co-expressed on monocytes. Current methods forisolating human CD4+ cells from peripheral blood mononuclear cells inhigh purity requires a pre-enrichment step to remove contaminatingmonocytes either with magnetic particles or by adherence to plasticplates. Thereafter, the CD4+ lymphocytes are isolated using anti-CD4coated magnetic particles.

As shown in FIG. 14, a monocyte-specific antibody recognizing themonocyte marker CD14, such as clone #63D3 (catalogue #367102; BioLegendInc., San Diego, Calif.), is conjugated to microbubbles and ananti-CD4-specific antibody, such as clone #SK3 (catalogue #344602;BioLegend Inc., San Diego, Calif.), is conjugated to nanomagneticparticles. With magnetibuoyant cell isolation the buoyant CD14/CD4double positive monocytes are lifted away from the CD4 single positive Tcells, which are captured to the walls of the tube with magnetic force.This results in a significant reduction in processing time and increasedthroughput can be realized.

Definitions:

In the context of the invention described above and in the claims below,the following terms will be understood to have the meanings ascribed. Inaddition to these terms, others are defined elsewhere in thespecification, as necessary. Unless otherwise expressly defined below orelsewhere in the specification, terms of art used in this specificationwill have their art-recognized meanings.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “about” refers to approximately a +/−10%variation from the stated value. It is to be understood that such avariation is always included in any given value provided herein, whetheror not it is specifically referred to.

An “analyte” refers to the substance to be detected, which may besuspected of being present in the sample (i.e., the biological sample).The analyte can be any substance for which there exists a naturallyoccurring specific binding partner or for which a specific bindingpartner can be prepared. Thus, an analyte is a substance that can bindto, or be bound by, one or more specific binding partners.

An “antibody” refers to a protein consisting of one or more polypeptidessubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. This term encompasses polyclonal antibodies,monoclonal antibodies, and antigen-binding antibody fragments, as wellas molecules engineered from immunoglobulin gene sequences thatspecifically bind an antigen of interest. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE,respectively. A typical immunoglobulin (antibody) structural unit isknown to comprise a tetramer. Each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” (about 25 kD)and one “heavy” chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms “variable lightchain (VL)” and “variable heavy chain (VH)” refer to these light andheavy chains, respectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized antigen-binding antibody fragments produced bydigestion with various peptidases. Thus, for example, pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab′)₂, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab′)₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)₂ dimer into a Fab′ monomer. The Fab′ monomer is essentially a Fabwith part of the hinge region. While various antigen-binding antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments may be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, in the context of the invention the term “antibody” also includesantigen-binding antibody fragments either produced by the modificationof whole antibodies or synthesized de novo using recombinant DNAmethodologies. Antibodies include single chain antibodies (antibodiesthat exist as a single polypeptide chain), single chain Fv antibodies(sFv or scFv), in which a variable heavy and a variable light chain arejoined together (directly or through a peptide linker) to form acontinuous polypeptide. The single chain Fv antibody is a covalentlylinked VH-VL heterodimer that may be expressed from a nucleic acidincluding VH- and VL-encoding sequences either joined directly or joinedby a peptide-encoding linker. While the VH and VL are connected to eachas a single polypeptide chain, the VH and VL domains associatenon-covalently. The scFv antibodies and a number of other structuresconvert the naturally aggregated, but chemically separated, light andheavy polypeptide chains from an antibody V region into a molecule thatfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart.

A “binding partner” or “member” of a high affinity binding pair is amember of a binding pair, i.e., a pair of molecules wherein one of themolecules binds to the second molecule. Binding partners that bindspecifically are termed “specific binding partners.” A “high affinity”binding pair is one in which the members bind with high affinity. Inaddition to antigen and antibody binding partners commonly used inimmunoassays, other specific binding partners can include biotin andavidin (or streptavidin), carbohydrates and lectins, nucleic acids withcomplementary nucleotide sequences, ligand and receptor molecules,cofactors and enzymes, enzyme inhibitors and enzymes, and the like.Furthermore, specific binding partners can include partner(s) thatis/are analog(s) of the original specific binding partner, for example,an analyte-analog. Immunoreactive specific binding partners includeantigens, antigen fragments, antibodies (monoclonal and polyclonal) andantigen-binding antibody fragments.

A “biological sample” is a sample of biological material taken from apatient or subject, as well as samples taken from tissue culture ortissue culture supernatants or any other source that could contain theanalyte of interest. Biological samples include samples taken frombodily fluids and tissues (e.g., from a biopsy) or tissue preparations(e.g., tissue sections, homogenates, etc.). A “bodily fluid” is anyfluid obtained or derived from a subject suitable for use in accordancewith the invention. Such fluids include whole blood, blood fractionssuch as serum and plasma, urine, sweat, lymph, feces, ascites, seminalfluid, sputum, nipple aspirate, post-operative seroma, wound drainagefluid, saliva, synovial fluid, bone marrow, cerebrospinal fluid, nasalsecretions, amniotic fluid, bronchoalveolar lavage fluid, peripheralblood mononuclear cells, total white blood cells, lymph node cells,spleen cells, and tonsil cells.

The terms “e.g.,” “such as”, and like terms mean “for example”, and thusdo not limit the term or phrase they explain, whereas the term “i.e.,”and like terms mean “that is”, thus limiting the term or phrase itexplains.

As used herein, the term “epitope” or “epitopes,” or “epitopes ofinterest” refer to a site(s) on any molecule that is recognized and iscapable of binding to a complementary site(s) on its specific bindingpartner. The epitope-bearing molecule and specific binding partner arepart of a specific binding pair. For example, an epitope can be apolypeptide, protein, hapten, carbohydrate antigen (such as, but notlimited to, glycolipids, glycoproteins or lipopolysaccharides) orpolysaccharide and its specific binding partner, can be, but is notlimited to, an antibody, e.g., an autoantibody. Typically an epitope iscontained within a larger molecular framework (e.g., in the context ofan antigenic region of a protein, the epitope is the region or fragmentof the protein having the structure capable of being bound by anantibody reactive against that epitope) and refers to the preciseresidues known to contact the specific binding partner. As is known, itis possible for an antigen or antigenic fragment to contain more thanone epitope.

“Herein” means in the present application, including anything that maybe incorporated by reference.

The terms “including”, “comprising”, and variations thereof mean“including, but not necessarily limited to”. Thus, for example, thephrase “the composition includes a drug and carrier” means thecomposition includes the drug and the carrier, but may also include oneor more other unspecified components as well.

As used herein, “specific” or “specificity” in the context of aninteraction between members of a specific binding pair (e.g., an antigenand antibody that specifically binds such antigen) refers to theselective reactivity of the interaction. The phrase “specifically bindsto” and analogous terms refer to the ability of antibodies tospecifically bind to (e.g., preferentially react with) an antigen andnot specifically bind to other entities. Antibodies or antigen-bindingantibody fragments that specifically bind to a particular antigen can beidentified, for example, by diagnostic immunoassays (e.g.,radioimmunoassays (“RIA”) and enzyme-linked immunosorbent assays(“ELISAs”), surface plasmon resonance, or other techniques known tothose of skill in the art. In one embodiment, the term “specificallybinds” or “specifically reactive” indicates that the binding preference(e.g., affinity) for the target analyte is at least about 2-fold, morepreferably at least about 5-fold, 10-fold, 100-fold, 1,000-fold, amillion-fold or more over a non-specific target molecule (e.g., arandomly generated molecule lacking the specifically recognizedsite(s)).

The term “labeled” refers to a molecule (e.g., an antibody,nanoparticle, etc.) that is labeled with a detectable label or becomeslabeled with a detectable label during use. A “detectable label”includes any moiety that is detectable or that can be rendereddetectable. With reference to a labeled separable particle, a “directlabel” is a detectable label that is attached to or associated with,covalently or non-covalently, the particle, and an “indirect label” is adetectable label that specifically binds the particle. Thus, an indirectlabel includes a moiety that is the specific binding partner of a moietyof the detection agent. Biotin and avidin are examples of such moietiesthat can be employed, for example, by contacting a biotinylated antibodywith labeled avidin to produce an indirectly labeled antibody (and thuslabeled nanomagnetic particle). A “label” refers to a detectablecompound or composition, such as one that is conjugated directly orindirectly to a target-specific binding member. The label may itself bedetectable by itself (e.g., a Raman label, a radioisotope, a fluorescentlabel, etc.) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition that isdetectable.

A “microparticle” refers to a small particle that is recoverable by anysuitable process, e.g., magnetic separation or association,ultracentrifugation, etc. Microparticles typically have an averagediameter on the order of about 1 micron or less.

A “nanoparticle” refers to a small particle that is recoverable by anysuitable process, e.g., magnetic separation or association,ultracentrifugation, etc. Nanoparticles typically have an averagediameter on the order of about 500 nanometers (nm) or less, preferablyfrom about 20 nm to about 300 nm, or any size or size range within such1 nm-about 500 nm size range.

A “patentable” process, machine, or article of manufacture according tothe invention means that the subject matter satisfies all statutoryrequirements for patentability at the time the analysis is performed.For example, with regard to novelty, non-obviousness, or the like, iflater investigation reveals that one or more claims encompass one ormore embodiments that would negate novelty, non-obviousness, etc., theclaim(s), being limited by definition to “patentable” embodiments,specifically excludes the unpatentable embodiment(s). Also, the claimsappended hereto are to be interpreted both to provide the broadestreasonable scope, as well as to preserve their validity. Furthermore, ifone or more of the statutory requirements for patentability are amendedor if the standards change for assessing whether a particular statutoryrequirement for patentability is satisfied from the time thisapplication is filed or issues as a patent to a time the validity of oneor more of the appended claims is questioned, the claims are to beinterpreted in a way that (1) preserves their validity and (2) providesthe broadest reasonable interpretation under the circumstances.

A “plurality” means more than one.

The terms “separated”, “purified”, “isolated”, and the like mean thatone or more components of a sample or reaction mixture have beenphysically removed from, or diluted in the presence of, one or moreother components present in the mixture.

The term “species” is used herein in various contexts, e.g., aparticular target biomolecule species. In each context, the term refersto a population of chemically indistinct molecules of the sort referredin the particular context.

REFERENCES

1. Massart, R., IEEE Trans. Magn., v17(2), p1247-1248 (1981).

2. Schwertmann, U., Cornell, R. M., Iron Oxides in the Laboratory:Preparation and Characterization: VCH Publication (New York, N.Y.),ISBN:3527269916 (1991).

3. Wetzel, R., et al., Eur. J. Biochem: v104, p469-478 (1980).4. Hsu CH, Chen C, Irimia D, Toner M. Fast sorting of CD4+ T cells fromwhole blood using glass microbubbles. Technology (Singap World Sci).2015 March; 3(1):38-44.5. Liou Y R, Wang Y H, Lee C Y, Li PC. Buoyancy-activated cell sortingusing targeted biotinylated albumin microbubbles. PLoS One. 2015 May20;10(5).6. Shi G, Cui W, Mukthavaram R, Liu Y T, Simberg D. Binding andisolation of tumor cells in biological media with perfluorocarbonmicrobubbles. Methods. 2013 Dec. 1; 64(2):102-7.7. Shi G, Cui W, Benchimol M, Liu Y T, Mattrey R F, Mukthavaram R,Kesari S, Esener S C, Simberg D. Isolation of rare tumor cells fromblood cells with buoyant immuno-microbubbles. PLoS One. 2013;8(3).

8. Clarke, J., Braginski, A. I., v1, SQUID Handbook;ISBN#3-527-40229-2;(2004); Berlin:Wiley-VCH.

9. Miltenyi et al., Cytometry: v11, p231-238 (1990)10. Kevin R, et al. Magnetic particle detection (MPD) for in-vitrodosimetry, Biosensors and Bioelectronics Volume 43,15 May 2013, Pages88-93

All of the compositions, articles, devices, systems, and methodsdisclosed and claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions, articles, devices, systems, and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions, articles, devices, systems, and methods withoutdeparting from the spirit and scope of the invention. All suchvariations and equivalents apparent to those skilled in the art, whethernow existing or later developed, are deemed to be within the spirit andscope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications are herein incorporated by reference intheir entirety for all purposes and to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference in its entirety for any and all purposes.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims, which may also containeven further embodiments of the invention.

1. A targeted nanomagnetic particle, comprising: (a) a magnetic core particle; (b) a glass layer encapsulating the magnetic core particle; (c) a protein/polymer composite layer bound to the glass layer; and (d) a targeting moiety that comprises one member of a bioaffinity ligand pair bound to the protein/polymer composite layer. 2-12. (canceled)
 13. A method of making targeted nanomagnetic particles according to claim 1, comprising: (a) obtaining magnetic core particles having a plurality of diameters ranging from about 5 nm to about 500 nm, preferably from about 30 nm to about 300 nm; (b) encapsulating the magnetic core particles with a glass layer to form first encapsulated magnetic core particles, wherein the glass layer optionally has a thickness of about 1 nm to about 50 nm; (c) encapsulating the first encapsulated magnetic core particles in a protein/polymer composite layer to form second encapsulated magnetic core particles, wherein protein/polymer composite layer optionally has a thickness of about 5 nm to about 50 nm; and (d) binding, optionally covalently, targeting moieties to the second encapsulated magnetic core particles, wherein the targeting moieties comprise one member of a bioaffinity ligand pair, optionally wherein the targeting moieties are independently selected from the group consisting of antibodies, an antigen-binding antibody fragments, a cell surface receptor, ligand-binding extracellular domains of a cell surface receptor, nucleic acids, avidin, streptavidin, biotin, pharmaceutical compounds for purposes of targeted drug delivery; and, optionally, (e) binding, optionally covalently, a pharmaceutical compound or detectable label to the second encapsulated magnetic core particles.
 14. A method according to claim 13 wherein the magnetic core particles are peptized.
 15. A method of forming biomolecule/particle complexes, comprising binding targeted nanomagnetic particles according to claim 1 to biomolecules of interest that are directly or indirectly specifically reactive with the targeting moieties of the targeted nanomagnetic particles to form biomolecule/particle complexes.
 16. A method according to claim 15 wherein the binding occurs in vitro or in vivo.
 17. A method of using biomolecule/particle complexes, comprising: (a) contacting a biological sample known or suspected to contain a biomolecule of interest with targeted nanomagnetic particles according to claim 1 to form biomolecule/particle complexes; and (b) using a magnetic field to isolate the biomolecule/particle complexes.
 18. A method according to claim 17 wherein a magnetic column separation or high gradient magnetic separation (HGMS) method is used to isolate the biomolecule/particle complexes.
 19. A targeted nanomagnetic particle according to claim 1 that behaves as a stable colloid when mixed with complex liquids, optionally mammalian whole blood or a fraction of mammalian whole blood.
 20. (canceled)
 21. A targeted nanomagnetic particle according to claim 1 that shows no significant or deleterious change in its magnetic, bioaffinity, and particle size and targeting properties during storage over a period of up to 5 years.
 22. A method of separating a target biomolecule species from a biological sample, comprising: (a) in a reaction mixture, contacting a biological sample known or suspected to contain a biomolecule of interest with targeted nanomagnetic particles according to claim 1 to form biomolecule/particle complexes; and (b) using a magnetic field to isolate the biomolecule/particle complexes from the reaction mixture.
 23. A method of separating a plurality of target biomolecule species from a biological sample, comprising: (a) in a reaction mixture, contacting a biological sample known or suspected to contain first and second biomolecule species of interest with targeted nanomagnetic particles according to claim 1 that target the first biomolecule species of interest to form first target biomolecule/particle complexes and targeted buoyant microparticles that target the second biomolecule species of interest to form second target biomolecule/particle complexes; (b) using a magnetic field to isolate the first biomolecule/particle complexes from the reaction mixture; and (c) separating the second target biomolecule/particle complexes from the reaction mixture.
 24. A method according to claim 23 wherein the targeted nanomagnetic particles have a diameter ranging from about 5 nm to about 500 nm, preferably from about 30 nm to about 300 nm.
 25. A method according to claim 23 wherein the magnetic core particles of the targeted nanomagnetic particles comprise magnetite (Fe₃O₄) crystals, optionally wherein the magnetite crystals have a diameter ranging from about 5 nm to about 300 nm.
 26. A kit comprising a composition according to claim 23 in a container and optionally including instructions for use of the composition. 